Cellular fatty acids typically derive from uptake from the extracellular milieu and, to a lesser extent, de novo synthesis. Extracellular fatty acids must traverse the plasma membrane, after which they are activated to their CoA thioesters for subsequent metabolism. Both uptake and metabolism are rapid processes, and there has been considerable debate as to whether transport of fatty acids across the lipid bilayer of the plasma membrane proceeds by diffusion or requires transport proteins. One group of proteins proposed to translocate fatty acids is the six-member Fatty Acid Transport Protein (FATP) family. These proteins were designated as such because when overexpressed, host cells exhibited higher rates of accretion of radioactive or fluorescent fatty acids. However, one member of this family, FATP2, is identical to an enzyme with very long-chain acyl-CoA synthetase (ACSVL) activity. This enzyme (ACSVL1 or FATP2), was isolated using classical protein purification techniques. In fact, the six-member ACSVL protein family is identical to the six-member FATP family. We and others have established that all six proteins have acyl-CoA synthetase activity. It remains to be established whether they participate in the physical translocation process, or facilitate transport by trapping, as CoA derivatives, fatty acids that enter cells by diffusion. To characterize the biological functions of the ACSVLs, we are investigating the properties of the overexpressed proteins and the endogenous proteins. We observed that for many ACSVLs, the subcellular location of the overexpressed protein differs from that of the endogenous protein. Using RNA interference (siRNA), we knocked down expression of FATP4 (proposed name: ACSVL5) in Neuro2a cells. Activation of both long-chain (C16:0) and very long-chain fatty acids (C24:0) was decreased when FATP4 was depleted. Despite decreased enzyme activity, initial rates of uptake of [14C]C16:0 were not affected when FATP4 was depleted. In contrast, COS-1 cells overexpressing FATP4 showed enhanced [14C]C16:0 uptake. Neither endogenous (Neuro2a) nor overexpressed (COS-1) FATP4 was localized to plasma membrane under routine cell culture conditions, but rather were found in intracellular membrane compartments. We conclude that, in the cell lines studied, endogenous FATP4 does not function to translocate FA across the plasma membrane.
- Acyl-CoA synthetase
- Fatty acid
- Fatty acid transport protein
ASJC Scopus subject areas
- Cellular and Molecular Neuroscience